Bacterial, and other microbial interaction with host tissues can have beneficial (symbiotic) as well as deleterious (pathogenic) consequences. Invading microbes can be pathogenic. Consequently, host biological defense strategies have evolved to protect organisms from invasion by disease-causing microorganisms.
Microbial infection response systems include oxidative and non-oxidative mechanisms, utilizing compounds that are enzymatically synthesized in cells, as well as peptides that are single gene products. For example, anti-microbial peptides constitute an oxygen-independent host defense system found in organisms encompassing many taxonomic families. One major class of anti-microbial peptides is defined by conserved cysteine residue patterns and is termed defensins. For example, mammalian defensins, derived from skin, lung and intestine, exhibit antibiotic activity against a wide variety of pathogens, including Gram-positive and Gram-negative bacteria, fungi (e.g., Candida species) and viruses. See, for example, Porter et al., Infect. Immun. 65(6): 2396-401, 1997.
A major class of microbial peptides is called adhesins. Adhesins enable microbes to adhere to mammalian tissues, for example the oral, gastrointestinal, urogenital and respiratory tracts. For a pathogenic microorganism, this may be a primary route to colonization and/or invasion of the host. Conversely, natural microbial flora can adhere to host tissues and create beneficial symbiotic relationships such as nutritional benefits, and protection against colonization of pathogenic microbes. The host defenses involved in attracting and establishing beneficial microbial colonization, as opposed to pathogenic microbial colonization, are not well understood. However, host defenses that affect this balance may have anti-microbial, immunomodulatory, inflammatory, anti-inflammatory or other properties.
Thus, moieties having anti-microbial, adhesin-like, immunomodulatory, inflammatory, anti-inflammatory or other properties are sought. The present invention provides such polypeptides for these and other uses that should be apparent to those skilled in the art from the teachings herein.
Within one aspect, the present invention provides an isolated polynucleotide encoding a zsig63 polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 16 (Arg) to amino acid number 37 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 38 (Leu) to amino acid number 126 (Ala); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 127 (Pro) to amino acid number 219 (Gln); (d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 16 (Arg) to amino acid number 219 (Gln); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 219 (Gln). In one embodiment, the isolated polynucleotide disclosed above encodes a zsig63 polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 16 (Arg) to amino acid number 37 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 38 (Leu) to amino acid number 126 (Ala); (c) the amino acid sequence as shown in SEQ ID NO: 2 from amino acid number 127 (Pro) to amino acid number 219 (Gln); (d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 16 (Arg) to amino acid number 219 (Gln); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 219 (Gln). In another embodiment, the isolated polynucleotide disclosed above is selected from the group consisting of: (a) a polynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 173 to nucleotide 784; (b) a polynucleotide sequence as shown in SEQ ID NO:1 from nucleotide 128 to nucleotide 784; and (c) a polynucleotide sequence complementary to (a) or (b). In another embodiment, the isolated polynucleotide disclosed above comprises nucleotide 1 to nucleotide 657 of SEQ ID NO:3.
Within another aspect, the present invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a zsig63 polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:2 from amino acid number 16 (Arg) to amino acid number 219 (Gln); and a transcription terminator. In one embodiment, the expression vector disclosed above further comprises a secretory signal sequence operably linked to the DNA segment.
Within another aspect, the present invention provides a cultured cell into which has been introduced an expression vector as disclosed above, wherein the cell expresses a polypeptide encoded by the DNA segment.
Within another aspect, the present invention provides a DNA construct encoding a fusion protein, the DNA construct comprising: a first DNA segment encoding a polypeptide selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:2 from residue number 1 (Met) to residue number 15 (Ala); (b) the amino acid sequence of SEQ ID NO:2 from residue number 16 (Arg) to residue number 37 (Ser); (c) the amino acid sequence of SEQ ID NO:2 from residue number 38 (Leu) to residue number 126 (Ala); (d) the amino acid sequence of SEQ ID NO:2 from residue number 127 (Pro) to residue number 219 (Gln); and (e) the amino acid sequence of SEQ ID NO:2 from residue number 16 (Arg) to residue number 219 (Gln); and at least one other DNA segment encoding an additional polypeptide, wherein the first and other DNA segments are connected in-frame; and encode the fusion protein.
Within another aspect, the present invention provides a fusion protein produced by a method comprising: culturing a host cell into which has been introduced a vector comprising the following operably linked elements: (a) a transcriptional promoter; (b) a DNA construct encoding a fusion protein as disclosed above; and(c) a transcriptional terminator; and recovering the protein encoded by the DNA segment.
Within another aspect, the present invention provides an isolated zsig63 polypeptide comprising a sequence of amino acid residues that is at least 90% identical to an amino acid sequence selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 16 (Arg) to amino acid number 37 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 38 (Leu) to amino acid number 126 (Ala); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 127 (Pro) to amino acid number 219 (Gln); (d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 16 (Arg) to amino acid number 219 (Gln); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 219 (Gln). In one embodiment, the isolated polypeptide disclosed above comprises a sequence of amino acid residues selected from the group consisting of: (a) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 16 (Arg) to amino acid number 37 (Ser); (b) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 38 (Leu) to amino acid number 126 (Ala); (c) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 127 (Pro) to amino acid number 219 (Gln); (d) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 16 (Arg) to amino acid number 219 (Gln); and (e) the amino acid sequence as shown in SEQ ID NO:2 from amino acid number 1 (Met) to amino acid number 219 (Gln).
Within another aspect, the present invention provides a method of producing a zsig63 polypeptide comprising: culturing a cell as disclosed above; and isolating the zsig63 polypeptide produced by the cell.
Within another aspect, the present invention provides a method of detecting, in a test sample, the presence of an antagonist of zsig63 protein activity, comprising: transfecting a zsig63-responsive cell, with a reporter gene construct that is responsive to a zsig63-stimulated cellular pathway; and producing a zsig63 polypeptide by the method as disclosed above; and adding the zsig63 polypeptide to the cell, in the presence and absence of a test sample; and comparing levels of response to the zsig63 polypeptide, in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the antagonist of zsig63 activity in the test sample.
Within another aspect, the present invention provides a method of detecting, in a test sample, the presence of an agonist of zsig63 protein activity, comprising: transfecting a zsig63-responsive cell, with a reporter gene construct that is responsive to a zsig63-stimulated cellular pathway; and adding a test sample; and comparing levels of response in the presence and absence of the test sample, by a biological or biochemical assay; and determining from the comparison, the presence of the agonist of zsig63 activity in the test sample.
Within another aspect, the present invention provides a method of producing an antibody to zsig63 polypeptide comprising the following steps in order: inoculating an animal with a polypeptide selected from the group consisting of: (a) a polypeptide consisting of 9 to 204 amino acids, wherein the polypeptide consists of a contiguous sequence of amino acids in SEQ ID NO:2 from amino acid number 16 (Ala) to amino acid number 219 (Gln); and (b) a polypeptide according as disclosed above; (c) a polypeptide comprising amino acid number 16 (Arg) to 37 (Ser) of SEQ ID NO:2; (d) a polypeptide comprising amino acid number 38 (Leu) to 126 (Ala) of SEQ ID NO:2; (e) a polypeptide comprising amino acid number 127 (Pro) to 219 (Gln) of SEQ ID NO:2; (f) a polypeptide comprising amino acid number 16 (Arg) to amino acid number 219 (Gln) of SEQ ID NO:2; (g) a polypeptide comprising amino acid number 1 (Met) to amino acid number 219 (Gln) of SEQ ID NO:2; (h) a polypeptide comprising amino acid number 14 (Phe) to 19 (Arg) of SEQ ID NO:2; (i) a polypeptide comprising amino acid number 16 (Arg) to 21 (Phe) of SEQ ID NO:2; (j) a polypeptide comprising amino acid number 24 (Gly) to 29 (Asp) of SEQ ID NO:2; (k) a polypeptide comprising amino acid number 25 (Glu) to 30 (Asp) of SEQ ID NO:2; (l) a polypeptide comprising amino acid number 187Glu) to 192 (Glu) of SEQ ID NO:2; (m) a polypeptide comprising amino acid number 24 (Gly) to 33 (Pro) of SEQ ID NO:2; (n) a polypeptide comprising amino acid number 17 (Lys) to 33 (Pro) of SEQ ID NO:2; (o) a polypeptide comprising amino acid number 66 (Thr) to 73 (Pro) of SEQ ID NO:2; (p) a polypeptide comprising amino acid number 103 (Pro) to 108 (Gly) of SEQ ID NO:2; (q) a polypeptide comprising amino acid number 190 (Ala) to 197 (Glu) of SEQ ID NO:2; (r) a polypeptide comprising amino acid number 202 (Lys ) to 215 (Gly) of SEQ ID NO:2; and (s) a polypeptide comprising amino acid number 190 (Ala) to 215 (Glu) of SEQ ID NO:2; and wherein the polypeptide elicits an immune response in the animal to produce the antibody; and isolating the antibody from the animal.
Within another aspect, the present invention provides an antibody produced by the method as disclosed above, which binds to a zsig63 polypeptide. In one embodiment, the antibody disclosed above the antibody is a monoclonal antibody. Within another aspect, the present invention provides an antibody that binds to a polypeptide as disclosed above.
These and other aspects of the invention will become evident upon reference to the following detailed description of the invention.